Cytogenetic analysis of HER1/EGFR, HER2, HER3 and HER4 in 278 breast cancer patients

Abstract

Introduction: The HER (human EGFR related) family of receptor tyrosine kinases (HER1/EGFR (epidermal growth factor receptor)/c-erbB1, HER2/c-erbB2, HER3/c-erbB3 and HER4/c-erbB4) shares a high degree of structural and functional homology. It constitutes a complex network, coupling various extracellular ligands to intracellular signal transduction pathways resulting in receptor interaction and cross-activation. The most famous family member is HER2, which is a target in Herceptin therapy in metastatic status and also in adjuvant therapy of breast cancer in the event of dysregulation as a result of gene amplification and resulting protein overexpression. The HER2-related HER receptors have been shown to interact directly with HER2 receptors and thereby mutually affect their activity and subsequent malignant growth potential. However, the clinical outcome with regard to total HER receptor state remains largely unknown.

Methods: We investigated HER1-HER4, at both the DNA and the protein level, using fluorescence in situ hybridisation (FISH) probes targeted to all four receptor loci and also immunohistochemistry in tissue microarrays derived from 278 breast cancer patients.

Results: We retrospectively found HER3 gene amplification with a univariate negative impact on disease-free survival (hazard ratio 2.35, 95% confidence interval 1.08 to 5.11, p = 0.031), whereas HER4 amplification showed a positive trend in overall and disease-free survival. Protein expression revealed no additional information.

Conclusion: Overall, the simultaneous quantification of HER3 and HER4 receptor genes by means of FISH might enable the rendering of a more precise stratification of breast cancer patients by providing additional prognostic information. The continuation of explorative and prospective studies on all HER receptors will be required for an evaluation of their potential use for specific therapeutic targeting with respect to individualised therapy.

Figure 1

Anti-HER-immunostaining of mouse fibroblasts, stably transfected with HER1, HER2, HER3 or HER4. Chinese hamster ovary wild-type, Jurkat and the three additional transfected fibroblast cell lines served as negative controls (magnification of slide overview ×16, magnification of cutout ×400, small circle indicates magnified area).

Figure 2

Anti-HER1–HER4 FISH in breast cancer tissue of one patient (dual probes). HER1: red cen 7, green loc 7p11 (diploid); HER2: red cen 17, green loc 17q12 (amplified); HER3: green cen 12, red loc 12q13 (moderately amplified); HER4: green cen 2, red loc 2q33 (diploid); 4',6-diamidino-2-phenylindole core staining blue. Cen, centromere; loc, gene locus.

Figure 3

Distribution of HER1–4 FISH ratios. (a) Boxplots of HER1–HER4 FISH ratios (gene/centromere). (b) Magnified extract of (a) to demonstrate the different distribution pattern of ratios for each HER-family member.

Figure 4

Martingale residuals, plotted against HER1–HER4 FISH ratios, based on overall survival. FISH, fluorescence in situ hybridisation.

Figure 5

Kaplan–Meier curves of dichotomised variables, based on overall survival. Anti-HER1–HER4 immunohistochemistry (a1,b,c,d), anti-HER2 FISH (a2) and immunohistochemistry score 2+, stratified by FISH-amplified and non-amplified cases (a3). FISH, fluorescence in situ hybridisation.

Figure 6

Univariate analysis of HER1–HER4 FISH. Comparison of individually calculated hazard ratios (overall survival, HR_ind = exp [B_HERx × HERx continuous]), based on the hazard ratio in one-tenth intervals showing the rising or declining hazard level as a function of increasing fluorescence in situ hybridisation (FISH) ratio.

Figure 7

Multivariate analysis of dichotomised HER2 FISH with continuous HER1, HER3 or HER4 FISH. Individually calculated hazard ratios (overall survival, HRind = EXP [B_HER2 × HER2 dichotomised + B_HERx × HERx continuous]) for HER1 (a), HER3 (b) and HER4 (c) fluorescence in situ hybridisation, divided into HER2 amplified (ratio ≥ 1.6; filled circles) and HER2 non-amplified (ratio ≤ 1.5; open circles) patients.